The cytochrome P450 Cyp6t3 is not required for ecdysone biosynthesis in Drosophila melanogaster.
The cytochrome P450 Cyp6t3 is not required for ecdysone biosynthesis in Drosophila melanogaster.
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DOI：
10.17912/micropub.biology.000611
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发表时间：
2022
影响因子：
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通讯作者：
O'Connor, Michael B
中科院分区：
文献类型：
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作者：
Shimell, MaryJane;O'Connor, Michael B
类固醇激素20-羟基蜕皮酮(20E)对于昆虫的正常发育和中期转变的时机是必不可少的。因此，人们对确定和确定在幼虫阶段的主要内分泌器官前胸腺(PG)中调节20E产生的酶和信号通路的作用非常感兴趣。转录组学是一个强大的工具，它已经被用来识别在PG中上调或下调的新基因，这可能有助于20E的调控。推测的调控候选基因的其他功能特征通常包括qRT-PCR和/或RNAi介导的敲除PG中的候选mRNA，以评估该基因的表达是否在PG中表现出时间调节，以及它的表达是否对20E的产生和正确的发育过渡时间是必需的。虽然这些方法在识别20E产生的新调控因子方面已经被证明是卓有成效的，但表征假定的调控基因的无效表型是分配基因功能的金标准，因为RNAi已知会产生各种类型的“脱靶”效应。在这里，我们描述了果蝇Cyp6t3基因的遗传零突变表型。Cyp6t3最初在PG芯片筛选中被鉴定为差异调控基因，并基于RNAi介导的击倒表型和涉及E生物合成途径的各种中间化合物的补给实验，在E生物合成途径的“黑盒”步骤中分配了一个位置。相反，我们发现Crispr在Cyp6t3中产生的零突变是可行的，并且具有正常的发育时序。因此，我们得出结论，在典型的实验室生长条件下，Cyp6t3不是E产生所必需的，因此不是黑盒的必备酶成分。
The steroid hormone 20-hydroxyecdysone (20E) is essential for proper development and the timing of intermediary stage transitions in insects. As a result, there is intense interest in identifying and defining the roles of the enzymes and signaling pathways that regulate 20E production in the prothoracic gland (PG), the major endocrine organ of juvenile insect phases. Transcriptomics is one powerful tool that has been used to identify novel genes that are up- or down-regulated in the PG which may contribute to 20E regulation. Additional functional characterization of putative regulatory candidate genes typically involves qRT-PCR and/or RNAi mediated knockdown of the candidate mRNA in the PG to assess whether the gene’s expression shows temporal regulation in the PG and whether its expression is essential for proper 20E production and the correct timing of developmental transitions. While these methods have proved fruitful for identifying novel regulators of 20E production, characterizing the null phenotype of putative regulatory genes is the gold standard for assigning gene function since RNAi is known to generate various types of “off target” effects. Here we describe the genetic null mutant phenotype of the Drosophila melanogaster Cyp6t3 gene . Cyp6t3 was originally identified as a differentially regulated gene in a PG microarray screen and assigned a place in the “Black Box” step of the E biosynthetic pathway based on RNAi mediated knockdown phenotypes and rescue experiments involving feeding of various intermediate compounds of the E biosynthetic pathway. In contrast, we find that Crispr generated null mutations in Cyp6t3 are viable and have normal developmental timing. Therefore, we conclude that Cyp6t3 is not required for E production under typical lab growth conditions and therefore is not an obligate enzymatic component of the Black Box.